Investigating the Effect of Fennel and Cinnamon Combined Extract On
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Journal of J Appl Biotechnol Rep. 2019 Sep;6(3):96-100 Applied Biotechnology doi 10.29252/JABR.06.03.03 Reports Original Article Investigating the Effect of Fennel and Cinnamon Combined Extract on Spermatogenesis and Testis Tissues in Busulfan Induced Infertile Rats Raziyeh Chegini1, Pouriya Soleimani1, Morteza Sadeghi2, Rana Mohammad Yosef1, Fariba Zafari3* 1Student Research Committee, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran 2Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 3Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran Corresponding Author: Fariba Zafari, Assistant Professor of Anatomical Sciences, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran. Tel: +982833336003, Fax: +982833336006, Email: [email protected] Received January 13, 2019; Accepted May 21, 2019; Online Published September 11, 2019 Abstract Introduction: Busulfan is one of the common cancer treatment drugs with infertility side effects. Fennel and cinnamon are two medicinal plants with fertility enhancement properties. The aim of this study was to investigate the effects of fennel and cinnamon on busulfan induced infertile rats. Materials and Methods: Forty male Wistar rats were divided into four groups including: sham group: healthy rats without intervention, control group: Busulfan treated rats, fennel group: busulfan and fennel extract treated, fennel and cinnamon group: busulfan, fennel and cinnamon extract treatment. Testicular tissues were sampled and the testicular physical parameters and spermatogenesis level were evaluated by H & E staining and optical microscopy imaging. Results: The biggest and the smallest testis lengths were observed in cinnamon + fennel and fennel groups respectively (P < 0.05). The highest and lowest sperm levels were observed in the cinnamon + fennel group and fennel group respectively (P < 0.001). The total average of reproductive cells was the most in the cinnamon + fennel group (208.88) and had the least level in the control group (81.2). Conclusions: The combined extract of fennel and cinnamon significantly protect the testicular tissues against infertility effect of busulfan. However, the fennel extract alone increased the effect of busulfan in rats. Keywords: Fennel, Cinnamon, Infertility, Spermatogenesis, Testis Citation: Chegini R, Soleimani P, Sadeghi M, Mohammad Yosef R, Zafari F. Investigating the effect of fennel and cinnamon combined extract on spermatogenesis and testis tissues in busulfan induced infertile rats. J Appl Biotechnol Rep. 2019;6(3):96-100. doi:10.29252/JABR.06.03.03. Introduction (Foeniculum vulgare Mill) is an indigenous plant of Asia Spermatogenesis is a complex and multi-stage process. and Iran which is noticeable because of its health benefits Various factors such as stress, nutrition and certain drugs in traditional medicine.12 In recent research, fennel has been may affect this process and cause azoospermia or infertility. considered due to its antioxidant and anti-inflammatory One of the most common side effects of some chemotherapy properties.13,14 Also, a group of flavonoids called phytoestrogen drugs is men’s azoospermia.1-3 Busulfan is a leukemia exist in fennel seeds that have a competitive effect on binding treatment chemotherapy drug which is used before and to estrogen receptors. Actually, phytoestrogen maintains the after bone marrow transplantation. The administration of balance of unwanted reactions resulted from the changes in busulfan in male patients with malignant cancers can lead to feminine hormones before and after menopause in women.15 permanent or complete infertility.4,5 It seems that the disorder However, no studies have yet been done on the effects in spermatogenesis after busulfan treatment is due to the of fennel in spermatogenesis and infertility.16 Cinnamon busulfan alkylating effect on DNA, which mainly affects the (Cinnamomum zeylanicum) is a widely used alternative spermatogonia stem cells.6,7 So far, various studies have been medicine that is indigenous to India. It is an aromatic herbal conducted on the effects of busulfan on rat spermatogenesis plant that contains many antioxidant compounds such as during both fetal development8,9 and maturity stages.10,11 In all cinnamon aldehyde, terpenes, cinnamyl alcohol and safrole.17 of these studies, azoospermia and testis tissue damaging have Cinnamon is an extremely important plant in traditional been confirmed. Due to the high incidence of infertility in medicine and many properties, including fat reduction, patients undergoing chemotherapy, the use of new therapies antibacterial properties, antifungal properties and sexual has always been considered in different countries. Fennel enhancement properties have been reported to it.18,19 The Copyright © 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Effect of Fennel and Cinnamon Combined Extract on Spermatogenesis results from various studies confirm the fact that cinnamon measured and the specimens were then placed within a 48 extract has been effective in improving the function of the hour Buffin fixture. Then, the specimens were transferred reproductive system and increasing spermatogenesis in rats.20 to the microprocessor. After being overrun, the slices were Considering the increasing infertility in the males population stained with H & E staining. The ImageJ software was used and on the other hand, little research on the effect of fennel for the slides imaging. and cinnamon in the male reproductive system, in this study the synergistic effect of fennel and cinnamon extract and Statistical Analysis also the effect of fennel alone on the reproductive system of The results of the study were analyzed by SPSS and ANOVA. busulfan induced infertile rats has been investigated. The t test was used to evaluate the significant difference between groups and P ≤ 0.05 was considered as a significant Materials and Methods level for all calculations. Animal Preparation and Infertility Model For this study, 40 adult male Wistar rats (180-200 g) were Results prepared from the Razi Institute Center of Karaj, Iran. The Spermatogenesis Level of Studied Groups animals were administrated under standard laboratory Four weeks after treatments, the most spermatogonia number conditions according to the standard protocol at Qazvin was observed in the cinnamon + fennel group (73.19 ± 11.30) University of Medical Sciences Animal Breeding and and the lowest was observed in the fennel group (49.32 Development Center. The oligospermic model was performed ± 13.53). The difference between these two groups was by intravascular busulfan injection in two different dosages: significant (P < 0.05). The highest and lowest spermatocyte the first dosage of 25 mg/kg of busulfan and the second dosage numbers were observed in cinnamon + fennel (62.50 ± 13.70) of 15 mg/kg were administered 14 days later. and control group (13.67 ± 5.27) respectively. The highest and lowest spermatids numbers were observed in the cinnamon Animals Grouping and Treatment + fennel (73.19 ± 11.30) and control groups (15.7 ± 38.67) The rats were divided into 4 groups including sham group: respectively. Generally, the highest and lowest number of healthy rats without any intervention, control group: rats reproductive cells (spermatogonia, primary spermatocyte treated with busulfan in two doses of 25 mg/kg and 15 mg/ and spermatids) belonged to the fennel + cinnamon (208.88) kg with a 14 day interval, fennel group: Busulfan treated rats and control groups (81.2) (Table 1 and Figure 1). following with a fennel extract (100 mg/kg) intraperitoneal injection for a period of 5 days, and the fennel+cinnamon Testis Weight of the Studied Groups group: busulfan treated rats with a fennel (100 mg/kg) and In evaluation of the testis weight in the studied groups, the cinnamon (200 mg/kg) extract intraperitoneal injection for a testis weight in the fennel + cinnamon group was significantly period of 5 days. higher than the other groups. Among the groups, the highest testis weight (1.66 ± 0.17 g) belonged to the fennel + cinnamon Preparation of Fennel and Cinnamon Extract group and the lowest testis weight (1.22 ± 0.34 g) belonged Plants extraction was done by a pharmacologist at the Iranian to the fennel group, with a significant difference between the Research Institute of Pharmaceutical Sciences according to two groups (P < 0.05). The weight of the testis in the fennel the following protocol: 10 g of plant powder was added to 1 L group was even lower than the busulfan group (1.25± 0.89 g), of ethanol and extracted for 2 hours by ultrasonic apparatus. which indicates the negative effect of fennel on the testicular The extract was then placed in dark under laboratory (Table 2). conditions (25°C) for 24 hours. After filtration, the extract was concentrated by rotary evaporation (vacuum distillation) Testis Length and Width in Studied Groups at a temperature below 50°C. The extracts and fractions were In the evaluation of the size and length of the testis in the stored at -18°C until they were consumed. studied groups, the highest and lowest testis lengths was observed in fennel + cinnamon (2.32 ± 0.26 cm) and fennel Sampling and Assessments (1.56 ± 0.12 cm) groups respectively, and the difference According to the spermatogenesis period of rats which is between the two groups was statistically significant (P < 0.001). 4 weeks,21 the animal’s testis samples were prepared after The combined extract group had the best results regarding four weeks from the beginning of the treatment. For this the size of the testis compared to the control group. The testis purpose, after the anesthetic, the testis were removed and length in the fennel group (1.56 ± 0.12) was even lower than the parameters of weight, length and width of them were the busulfan treated group (2.17 ± 0.18).